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Projeto de investigação
Programa RABBIT - Functional characterization and design of protein nanocages
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Condensation and Protection of DNA by the Myxococcus xanthus Encapsulin
Publication . Almeida, Ana V.; Carvalho, Ana J.; Calmeiro, Tomás; Jones, Nykola C.; Hoffmann, Søren V.; Fortunato, Elvira; Pereira, Alice S.; Tavares, Pedro; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; CENIMAT-i3N - Centro de Investigação de Materiais (Lab. Associado I3N); DCM - Departamento de Ciência dos Materiais; MDPI - Multidisciplinary Digital Publishing Institute
Encapsulins are protein nanocages capable of harboring smaller proteins (cargo proteins) within their cavity. The function of the encapsulin systems is related to the encapsulated cargo proteins. The Myxococcus xanthus encapsulin (EncA) naturally encapsulates ferritin-like proteins EncB and EncC as cargo, resulting in a large iron storage nanocompartment, able to accommodate up to 30,000 iron atoms per shell. In the present manuscript we describe the binding and protection of circular double stranded DNA (pUC19) by EncA using electrophoretic mobility shift assays (EMSA), atomic force microscopy (AFM), and DNase protection assays. EncA binds pUC19 with an apparent dissociation constant of 0.3 ± 0.1 µM and a Hill coefficient of 1.4 ± 0.1, while EncC alone showed no interaction with DNA. Accordingly, the EncAC complex displayed a similar DNA binding capacity as the EncA protein. The data suggest that initially, EncA converts the plasmid DNA from a supercoiled to a more relaxed form with a beads-on-a-string morphology. At higher concentrations, EncA self-aggregates, condensing the DNA. This process physically protects DNA from enzymatic digestion by DNase I. The secondary structure and thermal stability of EncA and the EncA−pUC19 complex were evaluated using synchrotron radiation circular dichroism (SRCD) spectroscopy. The overall secondary structure of EncA is maintained upon interaction with pUC19 while the melting temperature of the protein (Tm) slightly increased from 76 ± 1 °C to 79 ± 1 °C. Our work reports, for the first time, the in vitro capacity of an encapsulin shell to interact and protect plasmid DNA similarly to other protein nanocages that may be relevant in vivo.
Functional Characterization and Design of Protein Nanocages
Publication . Almeida, Ana Viana de; Pereira, Maria Alice; Tavares, Pedro
Compartmentalization is an essential cellular mechanism that allows cells to create and
organize controlled microenvironments for specific metabolic pathways, increase their
reaction rate and/or protect the cell from the harmful effect of substrates or products. Due to
the lack of organelles, prokaryotes produce protein-based compartments by protomer selfassembly.
Encapsulins, one of the bacterial nanocompartments most recently described, are
protein nanocages with the ability of sequestering other smaller proteins (cargo proteins)
within their cavity. The physiological function of encapsulins seems to be determined by the
type of cargo proteins encapsulated. Myxococcus xanthus encapsulin (EncA) is constituted by
180 protomers assembled into a 32 nm wide cage protein. This protein naturally encapsulates
two ferritin-like proteins (EncB and EncC) and a third protein with no predicted activity. The
encapsulation of cargo proteins and the characterization of the complex are described in this
thesis. No major structural changes in EncA were detected upon cargo encapsulation but the
assembly of EncC is shown to be iron-dependent. The function of the shell protein in
stabilizing and protecting cargo proteins from thermal denaturation is also demonstrated.
Additionally, a novel function of binding and protection of circular double stranded plasmid
DNA (pUC19) by EncA was discovered and characterized. Finally, the intrinsic ability of EncA
to mineralize iron was observed and described as similar to L-chain ferritins. The
encapsulation of EncC or EncB within EncA renders the complexes into a ferritin-like catalytic
active system. Differences in the chemical nature of the mineral core formed in the presence of
molecular oxygen and hydrogen peroxide were probed, as well as the detection of ferric
intermediates in EncC during the ferroxidation and iron mineralization reactions using
Mössbauer spectroscopy.
Dps, or DNA-binding protein from starved cells, is another bacterial
nanocompartment composed of 12 monomers assembled into a cube-like cage protein ~ 9 nm
wide. These proteins are known to bind and protect DNA and to accumulate ferric iron in their
cavity, protecting the cell from reactive oxygen species. However, the mechanism by which
labile iron returns to the cellular medium is still poorly understood. As such, the second part
of this thesis describes the iron release mechanism from the Dps of Marinobacter
hydrocarbonoclasticus while using WrbA, a flavoprotein, as an electron-transfer partner, NADH
as an electron donor and two types of iron acceptors: an inorganic compound (1,10-
phenanthroline) and a metalloprotein (rubredoxin). Although in aerobic conditions the iron is
only released when an iron chelator is present, labile iron is released into the solvent in
anaerobic condition. However, the presence of a chelator increases the rate of release.
Rubredoxin was proven to be suitable as a putative biological partner for the iron release from
Dps with faster iron release than in the presence of 1,10-phenanthroline.
The Conformation of the N-Terminal Tails of Deinococcus grandis Dps Is Modulated by the Ionic Strength
Publication . Guerra, João P. L.; Blanchet, Clement E.; Vieira, Bruno J. C.; Almeida, Ana V.; Waerenborgh, João C.; Jones, Nykola C.; Hoffmann, Søren V.; Tavares, Pedro; Pereira, Alice S.; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; MDPI - Multidisciplinary Digital Publishing Institute
DNA-binding proteins from starved cells (Dps) are homododecameric nanocages, with N-and C-terminal tail extensions of variable length and amino acid composition. They accumulate iron in the form of a ferrihydrite mineral core and are capable of binding to and compacting DNA, forming low-and high-order condensates. This dual activity is designed to protect DNA from oxidative stress, resulting from Fenton chemistry or radiation exposure. In most Dps proteins, the DNA-binding properties stem from the N-terminal tail extensions. We explored the structural characteristics of a Dps from Deinococcus grandis that exhibits an atypically long N-terminal tail composed of 52 residues and probed the impact of the ionic strength on protein conformation using size exclusion chromatography, dynamic light scattering, synchrotron radiation circular dichroism and small-angle X-ray scattering. A novel high-spin ferrous iron-binding site was identified in the N-terminal tails, using Mössbauer spectroscopy. Our data reveals that the N-terminal tails are structurally dynamic and alter between compact and extended conformations, depending on the ionic strength of the buffer. This prompts the search for other physiologically relevant modulators of tail conformation and hints that the DNA-binding properties of Dps proteins may be affected by external factors.
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Fundação para a Ciência e a Tecnologia
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COVID/BD/152498/2022